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Regulation of the anaphase-promoting complex by the COP9 signalosome
Cell Cycle
Abstract and Figures
The COP9 complex (signalosome) is a known regulator of the proteasome/ubiquitin pathway. Furthermore it regulates the activity of the cullin-RING ligase (CRL) families of ubiquitin E3-complexes. Besides the CRL family, the anaphase-promoting complex (APC/C) is a major regulator of the cell cycle. To investigate a possible connection between both complexes we assessed interacting partners of COP9 using an in vivo protein-protein interaction assay. Hereby, we were able to show for the first time that CSN2, a subunit of the COP9 signalosome, interacts physically with APC/C. Furthermore, we detected a functional influence of the COP9 complex regarding the stability of several targets of the APC/C. Consistent with these data we showed a genetic instability of cells overexpressing CSN2.
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... NODE_52_12 functions as a COP9 signalosome (CSN) complex. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via ubiquitin degradation and signal transduction pathways 42,43 . Brockway et al. 44 demonstrated that deficiency of the csn gene resulted in an inability to activate the MAPK pathway, which interrupted proliferation of Caenorhabditis elegans. ...
Clonostachys chloroleuca 67-1 (formerly C. rosea 67-1) is a promising mycoparasite with great potential for controlling various plant fungal diseases. The mitogen-activated protein kinase (MAPK)-encoding gene Crmapk is of great importance to the mycoparasitism and biocontrol activities of C. chloroleuca. To investigate the molecular mechanisms underlying the role of Crmapk in mycoparasitism, a high-quality yeast two hybrid (Y2H) library of C. chloroleuca 67-1 was constructed, and proteins interacting with Crmapk were characterised. The library contained 1.6 × 10⁷ independent clones with a recombination rate of 96%, and most inserted fragments were > 1 kb. The pGBKT7-Crmapk bait vector with no self-activation or toxicity to yeast cells was used to screen interacting proteins from the Y2H library, resulting in 60 candidates, many linked to metabolism, cellular processes and signal transduction. Combined bioinformatics and transcriptome analyses of C. chloroleuca 67-1 and ΔCrmapk mutant mycoparasitising Sclerotinia sclerotiorum sclerotia, 41 differentially expressed genes were identified, which might be the targets of the Fus3/Kss1-MAPK pathway. The results provide a profile of potential protein interactions associated with MAPK enzymes in mycoparasites, and are of great significance for understanding the mechanisms of Crmapk regulating C. chloroleuca mycoparasitism.
... Knockdown of individual subunits of the CSN complex in Drosophila cells increases the G1 fraction (Kondo and Perrimon, 2011). The cell-cycle regulatory function of the CSN might be achieved through regulating the degradation of cell-cycle regulators, such as Rb, p27, and APC/C (Kob et al., 2009;Tomoda et al., 2002;Ullah et al., 2007;Yang et al., 2002). However, the G2/M-promoting function of COPS2 in ESCs seems to be independent of the whole CSN complex. ...
... Knockdown of individual subunits of the CSN complex in Drosophila cells increases the G1 fraction (Kondo and Perrimon, 2011). The cell-cycle regulatory function of the CSN might be achieved through regulating the degradation of cell-cycle regulators, such as Rb, p27, and APC/C (Kob et al., 2009;Tomoda et al., 2002;Ullah et al., 2007;Yang et al., 2002). However, the G2/M-promoting function of COPS2 in ESCs seems to be independent of the whole CSN complex. ...
Proper regulation of the cell cycle is essential to safeguard the genomic integrity of embryonic stem cells (ESCs) while maintaining the fast proliferation rate. The pluripotency factor OCT4 has been shown to inhibit CDK1 activation, thus preventing mitotic entry and facilitating the maintenance of genomic integrity. Yet, how ESCs enter mitosis in the presence of OCT4 remains unclear. We previously reported that COPS2 promotes the progression through the G2/M phase of mouse ESCs. In this study, through co-immunoprecipitation and mass spectrometric analysis, we found that COPS2 interacts with OCT4 and CDK1. We further demonstrated that COPS2 stimulates the activity of CDK1/CYCLIN B only when OCT4 is present. Consistently, COPS2 promotes the G2/M transition only in the presence of OCT4 in HeLa cells. Mechanistically, COPS2 attenuates the interaction between OCT4 and CDK1 by sequestering OCT4 and forming a COPS2/CDK1 complex, thus blocking the inhibitory effect of OCT4 on CDK1 activation.
... In meiosis, APC/C-mediated degradation of Cyclin B is required for the metaphase-to-anaphase transition [43], moreover, the activity that ubiquitination of Cyclin B1 is involved in exit from MII [48]. It is well established that the CSN interacts physically with the APC/C and regulates several targets including Cyclin A and Cdc6 in human U2OS osteosarcoma cells [49]. When the CSN was down-regulated, SnoN and Cdc6 were increased, while Cyclin A decreased and Cyclin B remained unchanged. ...
The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.
... Noteworthy, Set8 degradation is mediated by two other ligases: CRL1 Skp2 and anaphase promoting complex/cyclosome (APC/ C) Cdh1 [114]. The latter has not yet been shown to be modified by Nedd8 but it is also affected by the CSN [115]. Notably, CRL4A CDT2 also targets p21 for degradation [116,117]. ...
The COP9 signalosome (CSN) is a platform for protein communication in eukaryotic cells. It has an intrinsic metalloprotease that removes the ubiquitin (Ub)-like protein Nedd8 from cullins. CSN-mediated deneddylation regulates culling-RING Ub ligases (CRLs) and controls ubiquitination of proteins involved in DNA damage response (DDR). CSN forms complexes with CRLs containing cullin 4 (CRL4s) which act on chromatin playing crucial roles in DNA repair, checkpoint control and chromatin remodeling. Furthermore, via associated kinases the CSN controls the stability of DDR effectors such as p53 and p27 and thereby the DDR outcome. DDR is a protection against cancer and deregulation of CSN function causes cancer making it an attractive pharmacological target. Here we review current knowledge on CSN function in DDR.
... Therefore, it appears that in U2OS and HeLa cells, celltype-specific mechanisms might be in place that restrict APC/C activity. Possibly, deubiquitinases like USP28 (Zhang et al., 2006; Bassermann et al., 2008), lack of APC/C-specific E2 enzymes, weakened APC/C-proteasome interaction (Kob et al., 2009), a defect in the downregulation of APC/C substrate mRNAs and/or the moderate strength of premature APC/C induction (SupplementaryFigure S6) could be responsible for the low effectiveness of APC/C activity in these cells. Remarkably, we found that APC/C activation in G2 is associated with the induction of a senescence-like phenotype. ...
The long-term cellular response to DNA damage is controlled by the tumor suppressor p53. It results in cell-cycle arrest followed by DNA repair and, depending on the degree of damage inflicted, premature senescence or apoptotic cell death. Here we show that in normal diploid fibroblasts the ubiquitin ligase anaphase-promoting complex or cyclosome (APC/C)-Cdh1 becomes prematurely activated in G2 as part of the sustained long-term but not the rapid short-term response to genotoxic stress and results in the degradation of numerous APC/C substrates. Using HCT116 somatic knockout cells we show that mechanistically premature APC/C activation depends on p53 and its transcriptional target p21 that mediates the signal through downregulation of the APC/C inhibitor Emi1. Cdc14B is dispensable in this setting but might function redundantly. Our data suggest an unexpected role for the APC/C in executing a part of the p53-dependent DNA damage response that leads to premature senescence.
Chez les plantes comme chez les animaux, la taille des organes est une caractéristique cruciale dépendant de différents processus que sont la prolifération, la croissance, la différentiation et la mort cellulaires. Ces processus sont très précisément contrôlés et beaucoup de leurs régulateurs clefs sont fortement conservés chez les eucaryotes. Parmi eux, TRANSLATIONNALLY CONTROLLED TUMOR PROTEIN (TCTP), une protéine régulée lors de sa traduction, est connue pour jouer un rôle essentiel dans le développement des organes. Ma thèse vise à préciser la voie d'action de TCTP sur la progression du cycle cellulaire chez Arabidopsis thaliana. CSN4, une sous-unité du complexe CONSTITUTIVE PHOTOMORPHOGENESIS 9 SIGNALOSOME (CSN), a été identifiée comme interacteur de TCTP. Le complexe CSN régule l'activité, l'assemblage et la stabilité des complexes CULLIN-RING UBIQUITIN LIGASES (CRL), une classe majeure d'E3-ubiquitine ligases. Les CRL sont donc impliqués dans la dégradation par la voie Ubiquitine/protéasome de nombreux substrats dont des régulateurs clefs du cycle cellulaire. J'ai tout d'abord caractérisé l'interaction physique entre AtTCTP et AtCSN4 et montré que celle-ci se fait en dehors du complexe CSN. J'ai ensuite caractérisé l'existence, chez les mutants csn4, d'un délai embryonnaire semblable, bien que nettement moins important, à celui observé chez les mutants tctp. Enfin, j'ai mis en évidence que des défauts d'expression de régulateurs clefs du cycle cellulaire : uniquement chez des régulateurs de la transition G1/S (CYCD7;1 et KRP6) pour les mutants tctp et beaucoup plus variés chez les mutants csn4. J'ai également mené une étude protéomique globale montrant un enrichissement de protéines impliquées notamment dans la traduction à la fois parmi celles surexprimées chez tctp et celles sous-exprimées chez csn4. Les travaux durant ma thèse apportent de nouvelles connaissances sur les mécanismes par lesquels TCTP régule la transition G1/S du cycle cellulaire en association avec CSN4 et ouvrent sur de nouvelles questions et perspectives notamment sur l'implication de régulateurs clefs du cycle comme CYCD7;1 et KRP6 ou le lien avec la régulation de la traduction des protéines.
The constitutive photomorphogenesis 9 signalosome (COP9 or CSN) is an evolutionarily conserved multiprotein complex found in plants and animals. Because of the homology between the COP9 signalosome and the 19S lid complex of the proteosome, COP9 has been postulated to play a role in regulating the degradation of polyubiquitinated proteins. Many tumor suppressor and oncogene products are regulated by ubiquitination- and proteosome-mediated protein degradation. Therefore, it is conceivable that COP9 plays a significant role in cancer, regulating processes relevant to carcinogenesis and cancer progression (e.g., cell cycle control, signal transduction and apoptosis). In mammalian cells, it consists of eight subunits (CSN1 to CSN8). The relevance and importance of some subunits of COP9 to cancer are emerging. However, the mechanistic regulation of each subunit in cancer remains unclear. Among the CSN subunits, CSN5 and CSN6 are the only two that each contain an MPN (Mpr1p and Pad1p N-terminal) domain. The deneddylation activity of an MPN domain toward cullin-RING ubiquitin ligases (CRL) may coordinate CRL-mediated ubiquitination activity. More recent evidence shows that CSN5 and CSN6 are implicated in ubiquitin-mediated proteolysis of important mediators in carcinogenesis and cancer progression. Here, we discuss the mechanisms by which some CSN subunits are involved in cancer to provide a much needed perspective regarding COP9 in cancer research, hoping that these insights will lay the groundwork for cancer intervention.
The interplay between ubiquitin (Ub) family modifiers creates a regulatory network of Ub family proteins which is essential for cell growth and differentiation. One of the best studied crosstalks between Ub family modifiers is the stimulation of ubiquitination by Nedd8 (neural precursor cell expressed developmentally down regulated 8) modification. The neddylation-deneddylation pathway controls the selective ubiquitination of important cellular regulators targeted for proteolysis by the Ub proteasome system (UPS). In this process the cullin scaffolds of cullin-RING Ub ligases (CRLs) are neddylated, which allosterically activates the transfer of Ub to substrates of the CRLs. A major reaction of the regulatory network is the removal of Nedd8 by the COP9 signalosome (CSN), which converts CRLs into an inactive state. The CSN is a conserved protein complex that interacts with CRLs and possesses an intrinsic metalloprotease with a Jab1/Pad1/MPN+ (JAMM) motif responsible for deneddylation.
In the present chapter we focus on the CSN-mediated deneddylation and its biological significance. We summarize latest developments on the mechanism of the CSN and its association with supercomplexes. In addition, data on the regulation of CSN-mediated deneddylation are described. Moreover, dysfunctions of the CSN and their implication in the pathogenesis of diseases are discussed.
Actin structures are involved in several biological processes and the disruption of actin polymerisation induces impaired motility of eukaryotic cells. Different factors are involved in regulation and maintenance of the cytoskeletal actin architecture. Here we show that S100A10 participates in the particular organisation of actin filaments. Down-regulation of S100A10 by specific siRNA triggered a disorganisation of filamentous actin structures without a reduction of the total cellular actin concentration. In contrast, the formation of cytoskeleton structures containing tubulin was unhindered in S100A10 depleted cells. Interestingly, the cellular distribution of annexin A2, an interaction partner of S100A10, was unaffected in S100A10 depleted cells. Cells lacking S100A10 showed an impaired migration activity and were unable to close a scratched wound. Our data provide first insights of S100A10 function as a regulator of the filamentous actin network.
SCF ubiquitin ligases control various processes by marking regulatory proteins for ubiquitin-dependent proteolysis. To illuminate
how SCF complexes are regulated, we sought proteins that interact with the human SCF component CUL1. The COP9 signalosome
(CSN), a suppressor of plant photomorphogenesis, associated with multiple cullins and promoted cleavage of the ubiquitin-like
protein NEDD8 fromSchizosaccharomyces pombe CUL1 in vivo and in vitro. Multiple NEDD8-modified proteins uniquely accumulated in CSN-deficientS. pombe cells. We propose that the broad spectrum of activities previously attributed to CSN subunits—including repression of photomorphogenesis,
activation of JUN, and activation of p27 nuclear export—underscores the importance of dynamic cycles of NEDD8 attachment and
removal in biological regulation.
The COP9 signalosome (CSN) is composed of eight distinct subunits and is highly homologous to the lid sub-complex of the 26S proteasome. CSN was initially defined as a repressor of photomorphogenesis in Arabidopsis, and it has now been found to participate in diverse cellular and developmental processes in various eukaryotic organisms. Recently, CSN was revealed to have a metalloprotease activity centered in the CSN5/Jab1 subunit, which removes the post-translational modification of a ubiquitin-like protein, Nedd8/Rub1, from the cullin component of SCF ubiquitin E3 ligase (i.e., de-neddylation). In addition, CSN is associated with de-ubiquitination activity and protein kinase activities capable of phosphorylating important signaling regulators. The involvement of CSN in a number of cellular and developmental processes has been attributed to its control over ubiquitin-proteasome-mediated protein degradation.
The nuclear localized, multi-subunit COP9 complex (or COP9 signalosome) is a key developmental modulator involved in repression of photomorphogenesis. In an effort to further define the molecular actions of the COP9 complex, a yeast two hybrid interactive screen was undertaken to identify proteins that could directly interact with one subunit of this complex, namely FUS6/COP11. This screen identified one specific interactive protein, AtS9, that is likely the Arabidopsis non-ATPase S9 (subunit 9) of the 19S regulatory complex from the 26S proteasome. AtS9 specifically interacts with FUS6/COP11 via the C-terminal domain of FUS6/COP11, which is distinct from the N-terminal domain necessary for FUS6/COP11 to interact with itself. As anticipated, AtS9 is not a member of the COP9 complex, but tightly associates with an ATPase subunit of the Arabidopsis 19S proteasome regulatory complex, AtS6A. Since all three proteins, FUS6/COP11, AtS9, and AtS6A, are present as complexed forms in vivo, the observed interaction implies that the COP9 complex may directly interact with the 19S regulatory complex of the 26S proteasome or other potential AtS9-containing complex. This notion is consistent with the parallel tissue-specific expression patterns and the similar, predominantly nuclear localization of both the COP9 complex and the AtS9 protein.
Annexin A5 is a Ca(2+)-binding protein which is involved in membrane organization and dynamics. As recent data suggest a role of annexin A5 in cancer we aimed to gain more insight into the biological function of endogenous annexin A5 and assessed its possible influence on proliferation and invasion capacity. We downregulated annexin A5 by RNA interference in HaCaT keratinocytes, squamous carcinoma cell line A431 as well as in a primary cell culture of a human oral carcinoma. Hereby, we detected reduced migration and invasion capacity of HaCaT cells which was even stronger in the oral carcinoma. To determine target genes of annexin A5 we used a metastasis specific microarray. Thereby, genes implicated in cell motility including S100A4, TIMP-3 and RHOC were observed to be regulated. These deregulations were confirmed by RT-PCR or western blots, respectively. These observations suggest that the invasion capacity, a main characteristic of tumors, is at least partially regulated by annexin A5 in oral carcinoma.
The anaphase-promoting complex/cyclosome (APC/C) controls the onset of anaphase by targeting securin for destruction. We report here the identification and characterization of a substrate of APC/C, RCS1, as a mitotic regulator that controls the metaphase-to-anaphase transition. We showed that the levels of RCS1 fluctuate in the cell cycle, peaking in mitosis and dropping drastically as cells exit into G1. Indeed, RCS1 is efficiently ubiquitinated by APC/C in vitro and degraded during mitotic exit in a Cdh1-dependent manner in vivo. APC/C recognizes a unique D-box at the N terminus of RCS1, as mutations of this D-box abolished ubiquitination in vitro and stabilized the mutant protein in vivo. RCS1 controls the timing of the anaphase onset, because the loss of RCS1 resulted in a faster progression from the metaphase to anaphase and accelerated degradation of securin and cyclin B. Biochemically, mitotic RCS1 associates with the NuRD chromatin-remodeling complex, and this RCS1 complex is likely involved in regulating gene expression or chromatin structure, which in turn may control anaphase onset. Our study uncovers a complex regulatory network for the metaphase-to-anaphase transition.
• HDAC1/2
• mitosis
• ubiquitin-dependent proteolysis
• Cdh1
The COP9 signalosome (CSN) is an evolutionarily conserved protein complex formed by eight subunits (CSN1 through CSN8). Deneddylating cullin family proteins is considered the bona fide function of the CSN. It has been proposed that the CSN regulates the assembly and disassembly of the cullin-based ubiquitin ligases via its deneddylation activity. Here we report that down-regulation of CSN8 by RNA interference destabilized differentially other CSN subunits and reduced the amount of CSN holo-complexes, leading to increases in neddylated cullin proteins and reduction of F-box protein Skp2 in HEK293 cells. Moreover, suppression of CSN8 enhanced the degradation of a proteasome surrogate substrate and cyclin kinase inhibitor p21(cip). Reduced transcript levels of cyclin kinase inhibitor p21(cip) and p27(kip) were also observed upon down-regulation of CSN8. These data suggest that the homeostatic level of CSN8/CSN suppresses proteasome proteolytic function and regulates transcription.
Cyclin B, a positive regulatory subunit of the cdc2 protein kinase complex, is synthesized across the cell cycle and then rapidly degraded at the end of mitosis. Degradation of cyclin B is triggered by increased levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquitin system, but the components responsible for the specificity and regulation of cyclin-ubiquitin ligation have not been identified. The formation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3). In this work we employed a fractionation approach to identify the components of a clam oocyte system responsible for specific ubiquitination of cyclin and to determine which components are regulated by cdc2. Experimental conditions were established under which a fusion protein containing an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractionation of M-phase extracts by DEAE-cellulose and high speed centrifugation yielded three fractions that were all required for cell cycle stage-specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E1. A second fraction contained a novel species of E2, termed E2-C, which acts in the ligation of ubiquitin to cyclin but not to other endogenous proteins. A third component is associated with particulate material. Whereas E2-C from either M-phase or interphase extracts is active, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein kinase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of about 30 min. These findings suggest that the particulate fraction may contain an E3 enzyme that acts on cyclin, as well as additional factors activated by cdc2.
The COP9 signalosome (CSN) is an eight-subunit protein complex that is found in all eukaryotes. Accumulating evidence indicates its diverse biological functions that are often linked to ubiquitin-mediated proteolysis. Here we applied an emerging mass spectrometry approach to gain insight into the structure of the CSN complex. Our results indicate that the catalytically active human complex, reconstituted in vitro, is composed of a single copy of each of the eight subunits. By forming a total of 35 subcomplexes, we are able to build a comprehensive interaction map that shows two symmetrical modules, Csn1/2/3/8 and Csn4/5/6/7, connected by interactions between Csn1-Csn6. Overall the stable modules and multiple subcomplexes observed here are in agreement with the "mini-CSN" complexes reported previously. This suggests that the propensity of the CSN complex to change and adapt its subunit composition might underlie its ability to perform multiple functions in vivo.